WO2013040436A2 - Modulateurs de transcription à médiation assurée par le gène esx et procédés associés - Google Patents

Modulateurs de transcription à médiation assurée par le gène esx et procédés associés Download PDF

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WO2013040436A2
WO2013040436A2 PCT/US2012/055543 US2012055543W WO2013040436A2 WO 2013040436 A2 WO2013040436 A2 WO 2013040436A2 US 2012055543 W US2012055543 W US 2012055543W WO 2013040436 A2 WO2013040436 A2 WO 2013040436A2
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esx
egfr
mediated transcription
transcription
cells
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PCT/US2012/055543
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WO2013040436A3 (fr
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Anna Mapp
Quintin PAN
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The Regents Of The University Of Michgian
The Ohio State University
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Priority to US14/344,069 priority Critical patent/US20150017156A1/en
Publication of WO2013040436A2 publication Critical patent/WO2013040436A2/fr
Publication of WO2013040436A3 publication Critical patent/WO2013040436A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • HNSCCs Head and neck squamous cell carcinomas
  • epithelial-restricted with serine box a member of the ETS transcription factor family
  • ESX serine box
  • shRNA-mediated knockdown of ESX in CAL27 cells resulted in decreased EGFR and Her2 protein levels and inhibited cell proliferation, invasion, and migration.
  • ESX knockdown dramatically dampened EGFR promoter activity demonstrating that ESX directly regulates EGFR expression.
  • the present invention provides small compounds capable of modulating ESX-mediated transcription and related methods of therapeutic and research use.
  • the present invention provides methods for treating conditions associated with aberrant EGFR expression with ESX-mediated transcription inhibitors.
  • R is selected from the group consisting of: , and m the
  • the methods are not limited to a particular disorder having elevated EGFR expression.
  • the disorder is a cancer or cancer related disorder.
  • the cancer is HNSCC.
  • the methods are not limited to a particular type of ESX-mediated transcription inhibitor.
  • the ESX-mediated transcription inhibitor is an isoxazolidine compound.
  • the methods are not limited to a particular isoxazolidine compound.
  • the isoxazolidine compound is represented by the following formula: including salts, esters and prodrugs thereof, wherein R is a functional group that mimics at least a portion of the eight amino acid (137-SWIIELLE-146) (SEQ ID NO:l) a-helical region in ESX.
  • R is a functional group that mimics the effect of Tryptophan 138 of ESX.
  • R is a functional group that mimics the formation of a hydrophobic surface along an amphipathic helix within amino acids 137-146 of ESX.
  • R is selected from the group consisting of:
  • biphenyl isoxazolidine enhanced the anti-proliferative effects of afatinib, gefitinib and lapatinib, three EGFR tyrosine kinase inhibitors. Indeed, the experiments indicate inhibition of ESX to reduce EGFR and Her2 levels as an approach to enhance the response rate of HNSCC patients to current anti-EGFR therapeutics.
  • the present invention provides methods for treating a subject having a disorder having elevated erbB2 expression.
  • the present invention is not limited to particular methods for treating disorders having elevated erbB2 expression.
  • the methods involve, for example, co-administering to the subject an effective amount of an ESX-mediated transcription inhibitor, and one or more agents known to target the activity and lifetime of the erbB2 oncoprotein. Any type of subject is contemplated for such methods (e.g., human, dog, cat, cow, ape, etc.).
  • R is a functional group that mimics at least a portion of the eight amino acid (137-SWIIELLE-146) (SEQ ID NO: l) a-helical region in ESX.
  • R is a functional group that mimics the effect of Tryptophan 138 of ESX.
  • R is a functional group that mimics the formation of a hydrophobic surface along an amphipathic helix within amino
  • the methods are not limited to particular agents known to target the activity and lifetime of the erbB2 oncoprotein.
  • the agent is a tyrosine kinase inhibitor (e.g., afatinib, gefitinib, erlotinib, lapatinib (see Example 5).
  • the present invention provides methods for identifying ESX- mediated transcription modulators, comprising, for example, providing i) host cells expressing ESX, a gene whose transcription is regulated by ESX, and ESX-mediated transcription coactivating compounds required for the ESX-mediated transcription of the gene of interest, and ii) a potential ESX-mediated transcription modulator, delivering to the host cells an effective amount of the potential ESX-mediated transcription modulator, and detecting changes in ESX-mediated transcription, wherein inhibition in ESX-mediated transcription indicates the potential ESX-mediated transcription modulator is an ESX- mediated transcription inhibitor, wherein enhancement in ESX-mediated transcription indicates the potential ESX-mediated transcription modulator is an ESX-mediated transcription enhancer.
  • CAL27/shRNA-ESX cells were harvested, and resuspended in serum-free medium. An aliquot (1 X 105 cells) of the prepared cell suspension was added to the top chamber and 10% FBS was added to the bottom chamber. After 24 hours, non-invading cells were gently removed from the interior of the inserts with a cottontipped swab. Invasive cells were visualized with fluorescence microscopy. A representative field for each experimental condition is presented. E. Cell migration. Cells were seeded and allowed to grow until confluence. Confluent monolayers were scratched using a sterile pipette tip, washed, and incubated in complete medium. A representative field for each experimental condition at 0 hour and 10 hours is presented.
  • FIG. 3 shows that biphenyl isoxazolidine reduces EGFR and inhibits cell proliferation, invasion, and migration in HNSCC.
  • IC50 was 46.8 ⁇ /L for CAL27 and 50.3 ⁇ /L for SCC25.
  • Cell migration was determined using the wound healing assay. Cells were seeded and allowed to grow until confluence. Confluent monolayers were treated with biphenyl isoxazolidine for 24 hours, scratched using a sterile pipette tip, washed, and incubated in complete medium. A representative field for each experimental condition at 0 hour and 24 hours is presented.
  • agents/therapies used may vary.
  • the appropriate dosage for co-administration can be readily determined by one skilled in the art.
  • the respective agents/therapies are administered at lower dosages than appropriate for their administration alone.
  • co- administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g. , toxic) agent(s).
  • Teroxirone Testolactone; Thiamiprine; Thioguanine; Thiotepa; Thymitaq; Tiazofurin;
  • MeCCNU N-(2-chloroethyl)-N'-(diethyl)ethylphosphonate-N-nit- rosourea
  • streptozotocin diacarbazine (DTIC); mitozolomide; temozolomide; thiotepa; mitomycin C;
  • Benign prostatic hyperplasia therapy agents e.g., Tamsulosin Hydrochloride
  • Tricyclic anti-depressant drugs e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline
  • non-tricyclic anti-depressant drugs e.g., sertraline, trazodone and citalopram
  • Ca ++ antagonists e.g., verapamil, nifedipine, nitrendipine and caroverine
  • Calmodulin inhibitors e.g., prenylamine, trifluoroperazine and clomipramine
  • Amphotericin B Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);
  • antihypertensive drugs e.g., reserpine
  • Thiol depleters e.g., buthionine and sulfoximine
  • Multiple Drug Resistance reducing agents such as Cremaphor EL.
  • Still other anticancer agents include, but are not limited to, annonaceous acetogenins; asimicin; rolliniastatin; guanacone, squamocin, bullatacin; squamotacin; taxanes; paclitaxel; gemcitabine;
  • anticancer agents 2'deoxyadenosine; Fludarabine-PC ; mitoxantrone; mitozolomide; Pentostatin; and Tomudex.
  • One particularly preferred class of anticancer agents are taxanes (e.g., paclitaxel and docetaxel).
  • Another important category of anticancer agent is annonaceous acetogenin.
  • instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Oilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al., 2002.
  • ESX-mediated transcription inhibitors with radiation therapy are not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal.
  • the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof.
  • the radiation is delivered to the animal using a linear accelerator.
  • the radiation is delivered using a gamma knife.
  • Radiotherapy any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects.
  • Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. Pat. No. 5,770,581 incorporated herein by reference in its entirety).
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.
  • any one or more of the ESX-mediated transcription inhibitors described herein are prepared by applying standard pharmaceutical manufacturing procedures. Such pharmaceutical formulations can be delivered to the subject by using delivery methods that are well-known in the pharmaceutical arts.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, wherein each preferably contains a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient is presented as a bolus, electuary, or paste, etc.
  • Molded tablets are made by molding in a suitable machine a mixture of the powdered compound (e.g. , active ingredient) moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • the aqueous phase of a cream base includes, for example, at least about 30% w/w of a polyhydric alcohol, i.e. , an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • a polyhydric alcohol i.e. , an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • oily phase emulsions of this invention are constituted from known ingredients in a known manner.
  • This phase typically comprises a lone emulsifier (otherwise known as an emulgent), it is also desirable in some embodiments for this phase to further comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • Formulations suitable for nasal administration wherein the carrier is a solid, include coarse powders having a particle size, for example, in the range of about 20 to about 500 microns which are administered in the manner in which snuff is taken, i.e. , by rapid inhalation (e.g. , forced) through the nasal passage from a container of the powder held close up to the nose.
  • suitable formulations wherein the carrier is a liquid for administration include, but are not limited to, nasal sprays, drops, or aerosols by nebulizer, an include aqueous or oily solutions of the agents.
  • the agents identified can be administered to subjects or individuals susceptible to or at risk of developing pathological growth of target cells and correlated conditions.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tissue sample is removed from the patient and the cells are assayed for sensitivity to the agent.
  • Therapeutic amounts are empirically determined and vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • in vivo administration is effected in one dose, continuously or intermittently throughout the course of treatment.
  • Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations are carried out with the dose level and pattern being selected by the treating physician.
  • Suitable dosage formulations and methods of administering the agents are readily determined by those of skill in the art.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • the effective amount may be less than when the agent is used alone.
  • the pharmaceutical compositions can be administered orally, intranasally, parenterally or by inhalation therapy, and may take the form of tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. They may also take the form of suspensions, solutions and emulsions of the active ingredient in aqueous or non-aqueous diluents, syrups, granulates or powders. In addition to an agent of the present invention, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
  • the therapeutic methods further comprise co-administering to the subject a therapeutic agent selected from the group consisting of an EGFR monoclonal antibody inhibitor (e.g., cetuximab, panitumumab, zalutumubab, nimotuzumab, matuzumab), a tyrosine kinase inhibitor (e.g., afatinib, gefitinib, erlotinib, lapatinib), and/or any therapeutic agents known/used for treating EGFR related disorders (e.g., cancer (e.g., HNSCC, lung cancer, colorectal cancer) (e.g., AP26113 (e.g., ARIAD Pharmaceuticals), potato
  • a therapeutic agent selected from the group consisting of an EGFR monoclonal antibody inhibitor (e.g., cetuximab, panitumumab, zalutumubab, nimotuzumab, matuzumab),
  • NMR spectroscopy suggests that W138 along with 1139, 1140, L142, and L143 form a hydrophobic surface along an amphipathic helix that interacts with Med23 (see, e.g., Asada et al, 2002).
  • the binding interaction between ESX and Med23 was shown to be disrupted with a small molecule a-helix mimic of ESX, wrenchnolol (Shimogawa et al, 2004).
  • Wrenchnolol decreased Her2 expression and inhibited cell proliferation of SKBR3 Her2-positive breast carcinoma cells (see, e.g., Shimogawa et al, 2004).
  • biphenyl isoxazolidine a novel a-helix ESX mimic, biphenyl isoxazolidine, was designed and synthesized to block the interaction between ESX-Med23 (see, e.g., Lee et al, 2009). Similar to wrenchnolol, biphenyl isoxazolidine decreased Her2 expression and inhibited cell proliferation of SKBR3 cells (see, e.g., Lee et al, 2009). These two studies showed that targeting the ESX-Med23 interaction is feasible and moreover, demonstrated that inhibition of transcription factor activation with small molecules is a novel and promising avenue for anti-cancer drug development.
  • biphenyl isoxazolidine can effectively suppress ESX transcriptional activity leading to a decrease in EGFR and Her2 levels and inhibition of cell invasion, motility, and proliferation.
  • Example 4 The concentration of biphenyl isoxazolidine needed to inhibit cell invasion and cell migration is much lower than required to inhibit cell proliferation. This observation argues that the EGFR gene dosage threshold to modulate cell invasion, migration, and proliferation is different; cell migration and invasion requires a higher level of EGFR than cell proliferation. Moreover, these results demonstrate that inhibition of cell invasion and migration mediated by biphenyl isoxazolidine is not due, for example, to a general decrease in cell viability but due, for example, to dampening of signal transduction pathways specific to cell morphology and movement. EGFR and Her2 are well recognized as regulators of cell invasion, migration, and proliferation. Taken together, these results show that biphenyl isoxazolidine can effectively suppress ESX transcriptional activity leading to a decrease in EGFR and Her2 levels and inhibition of cell invasion,
  • EGFR inhibitors such as cetuximab, a humanized anti-EGFR antibody, or TKIs, such as gefitinib, lapatinib, and erlotinib
  • TKIs such as gefitinib, lapatinib, and erlotinib
  • CAL27 and SCC25 cells were treated with gefitinib, an EGFR TKI, or lapatinib, a dual EGFR/Her2 TKI, at various concentrations with and without an IC50 dose of biphenyl isoxazolidine (Figure 4).
  • Single agent gefitinib and lapatinib inhibited the proliferation of CAL27 and SCC25 cells after 24 hours of treatment.
  • the IC50 was 26.3 ⁇ /L for gefitinib and 11.8 ⁇ /L for lapatinib in CAL27 cells.
  • the erbB2 protein is a trans-membrane tyrosine kinase that is over expressed in approximately one quarter of breast cancers (see, e.g., Slamon, et al., 1989), where it has been shown to drive an aggressive phenotype marked by more rapid metastasis and shorter life expectancy than breast cancers that do not over-express erbB2 (see, e.g., Yarden, 2000; Slamon, 1987). Furthermore, erbB2 over-expressing (erbB2+) cancer cells are known to undergo growth arrest and cell death if erbB2 expression is suppressed (see, e.g., Menendez, 2004).
  • erbB2 overexpression can be seen in the variety of existing treatments designed to suppress erbB2 signaling, including antibodies that target the protein's extracellular domain (see, e.g., Nahta, 2007) and tyrosine kinase inhibitors which target the protein's ability trans-phosphorylate other members of the erbB family and initiate cell survival and proliferation programs (Figure 5) (see, e.g., Xia, 2005).
  • Hsp90 part of a chaperone complex that maintains erbB2 stability and assists in membrane localization
  • the natural product geldanamycin reduces cellular erbB2 levels by binding to Hsp90 and inhibiting its function ( Figure 5) but its toxicity prevents its use as a therapeutic agent (see, e.g., Roe, 1999; Isaacs, 2003).
  • biphenyl isoxazoline:geldanamycin combination was 5: 1, and this combination is also synergistic as defined by the multiplicative additivity or Bliss model ( Figure 6c) (see, e.g., Berenbaum, 1989; Borisy, 2003). This degree of synergy increased proliferation from combination treatment (Supporting Figure S7b-c).
  • the combination of geldanamycin and biphenyl isoxazoline concomitantly produced an 85% drop in erbB2 levels (Supporting Figure S7a).
  • This example provides materials and methods.
  • Isoxazolidine il was prepared as described previously (see, e.g., Lee,
  • Lapatinib ditosylate and erlotinib were purchased from AK scientific, and
  • geldanamycin was a generous gift. The identity and purity of all compounds were verified by NMR analysis and HPLC. Antibodies were purchased from Santa Cruz Biotechnology. Absorbance data was collected on a Tecan GENios Pro.
  • Isobolograms were generated by computing dose fractions directly from the ICsos.
  • Dose fraction is defined as the dose of one component in a combination required to exert a given effect (usually ICso, as in this case) divided by the dose of that component in isolation required to exert the same effect.
  • each combination reported has two dose fraction measurements (eg. dose fraction il and dose fraction lapatinib) that define the combinations x/y coordinates on the isobologram.
  • SkBr3 and IMR90 cells were purchase from ATCC and cultured in RPMI 1640 (SkBr3) or DMEM (IMR90) with 10% FBS and no antibiotics.
  • RPMI 1640 SkBr3
  • DMEM DMEM
  • FBS FBS
  • no antibiotics FBS
  • cells were plated at 3000 cells per well in 96 well plates. After adhering overnight, media was changed to 2.5% FBS and compound (as a solution in DMSO) was added. New media and compound were added for each additional day of treatment (2 days in the case of lapatinib and 3 days in the case of geldanamycin). The day after the final treatment, cell viability was measured using WST-1 reagent (Roche) in accordance with the manufacturer's instructions.
  • ErbB2 and p-erbB2 ELISA ELISA assays were performed based on those published elsewhere. [4] In brief, SkBr3 cells were plated in 10% FBS at 15000 cells per well in 24 well plates. After adhering overnight, media was changed to 2.5% FBS and compound was added. After 24 hours, media was removed carefully and cells were fixed and permeabilized with cold (-20° C) methanol. The cells were then washed twice with TBST, blocked for 2 hours at room temperature with superblock (TBST solution from Pierce), incubated with the primary antibody overnight at 4° C (as a 1:500 solution in superblock).
  • Cells were then washed twice more with TBST and incubate with secondary antibody for 2 hours at room temperature (as a 1 :1000 solution in superblock). After being washed 3 times with TBST, Slow TMB (Pierce) was added to measure antibody levels in accordance with the manufacturer's instructions and the absorbance at 370 nm was measured. The cells were washed 3 more times with TBST and total protein levels were measured at 560 nm using BCA reagent (Pierce).
  • ErbB2 and p- erbB2 levels were normalized to total protein concentration by calculating (A370- A370blank)/(A560-A560 blank), where the A370 blank was from TMB treated cells not treated with a primary antibody, and the A560 blank was BCA reagent in wells that did not have cells plated in them. The resulting ratios were then normalized to cells treated with DMSO.
  • ErbB2 and ErbB3 associates with resistance and epidermal growth factor receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells.
  • EGFR epidermal growth factor receptor

Abstract

La présente invention concerne la régulation de gènes. En particulier, la présente invention concerne de petits composés pouvant moduler la transcription à médiation assurée par le gène ESX, et des procédés associés d'utilisation à des fins thérapeutiques et de recherche. En outre, la présente invention concerne des procédés pour traiter des états associés à l'expression aberrante du récepteur EGFR par des modulateurs de transcription à médiation assurée par le gène ESX (par exemple, des inhibiteurs de transcription à médiation assurée par le gène ESX)
PCT/US2012/055543 2011-09-16 2012-09-14 Modulateurs de transcription à médiation assurée par le gène esx et procédés associés WO2013040436A2 (fr)

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US10981903B2 (en) 2011-11-17 2021-04-20 Dana-Farber Cancer Institute, Inc. Inhibitors of c-Jun-N-terminal kinase (JNK)
US10144730B2 (en) 2011-11-17 2018-12-04 Dana-Farber Cancer Institute, Inc. Inhibitors of c-Jun-N-terminal kinase (JNK)
US10787436B2 (en) 2012-10-18 2020-09-29 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinase 7 (CDK7)
US10112927B2 (en) 2012-10-18 2018-10-30 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinase 7 (CDK7)
USRE48175E1 (en) 2012-10-19 2020-08-25 Dana-Farber Cancer Institute, Inc. Hydrophobically tagged small molecules as inducers of protein degradation
US9758522B2 (en) 2012-10-19 2017-09-12 Dana-Farber Cancer Institute, Inc. Hydrophobically tagged small molecules as inducers of protein degradation
US10000483B2 (en) 2012-10-19 2018-06-19 Dana-Farber Cancer Institute, Inc. Bone marrow on X chromosome kinase (BMX) inhibitors and uses thereof
US11040957B2 (en) 2013-10-18 2021-06-22 Dana-Farber Cancer Institute, Inc. Heteroaromatic compounds useful for the treatment of proliferative diseases
US10906889B2 (en) 2013-10-18 2021-02-02 Dana-Farber Cancer Institute, Inc. Polycyclic inhibitors of cyclin-dependent kinase 7 (CDK7)
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WO2015103317A1 (fr) * 2013-12-30 2015-07-09 Lifesci Pharmaceuticals, Inc. Composés inhibiteurs thérapeutiques
US11021463B2 (en) 2013-12-30 2021-06-01 Attune Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US10259803B2 (en) 2013-12-30 2019-04-16 Lifesci Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US10266515B2 (en) 2013-12-30 2019-04-23 Lifesci Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US10017477B2 (en) 2014-04-23 2018-07-10 Dana-Farber Cancer Institute, Inc. Janus kinase inhibitors and uses thereof
US9862688B2 (en) 2014-04-23 2018-01-09 Dana-Farber Cancer Institute, Inc. Hydrophobically tagged janus kinase inhibitors and uses thereof
US10870651B2 (en) 2014-12-23 2020-12-22 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinase 7 (CDK7)
US11325910B2 (en) 2015-03-27 2022-05-10 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinases
US10550121B2 (en) 2015-03-27 2020-02-04 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinases
AU2016276963B2 (en) * 2015-06-12 2021-05-06 Dana-Farber Cancer Institute, Inc. Combination therapy of transcription inhibitors and kinase inhibitors
US10702527B2 (en) 2015-06-12 2020-07-07 Dana-Farber Cancer Institute, Inc. Combination therapy of transcription inhibitors and kinase inhibitors
AU2016276963C1 (en) * 2015-06-12 2021-08-05 Dana-Farber Cancer Institute, Inc. Combination therapy of transcription inhibitors and kinase inhibitors
WO2016201370A1 (fr) * 2015-06-12 2016-12-15 Dana-Farber Cancer Institute, Inc. Thérapie d'association utilisant des inhibiteurs de transcription et des inhibiteurs de kinases
US10023557B2 (en) 2015-07-01 2018-07-17 Lifesci Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US10308637B2 (en) 2015-07-01 2019-06-04 Lifesci Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US11142507B2 (en) 2015-09-09 2021-10-12 Dana-Farber Cancer Institute, Inc. Inhibitors of cyclin-dependent kinases
US10781200B2 (en) 2016-07-11 2020-09-22 Attune Pharmaceuticals, Inc. Therapeutic inhibitory compounds
US10301284B2 (en) 2016-07-11 2019-05-28 Lifesci Pharmaceuticals, Inc. Therapeutic inhibitory compounds

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